Stimulation by ATP of [3H]dopamine binding to synaptic membrane fractions of canine caudate nucleus

The rate of [³H]dopamine binding to crude synaptic membranes from canine caudate nucleus was considerably increased by 2 mM ATP, 5′-adenylylimidodiphosphate and GTP or by 1 mM 5′-guanylyl-imidodiphosphate, while strongly inhibited by 2 mM ADP and GDP. Half maximal concentrations of [³H]dopamine to bind to the membranes were 1.11 × 10^?7M and 8.75 × 10^?6M in the absence of 4 mM ATP, indicating a negative cooperativity of the dopamine receptor, and 9.25 × 10^?7 M in its presence. Hill coefficient was increased from 0.70 to 1.04 by addition of 4 mM ATP. The optimal concentration of ATP for [³H]dopamine binding was in the range of 0.5 to 5 mM.     

Polyphosphate anions increase the activity of bovine spleen cathepsin D

Bovine spleen cathepsin D is activated by polyphosphate anions when bovine serum albumin is used as substrate at pH 4.6. In the presence of ATP at 10 mM, the catheptic activity at this pH is enhanced as high as 17 times over the control. Similar activating effects were observed, though to varying degrees, with sodium tripolyphosphate, nucleotides, nucleotide analogues, CoA, polyU and yeast RNA. The possible mechanism and biological significance of the activation were discussed with regard to the intralysosomal polyanionic substance.     

Concanavalin A binding to brain particulate preparations and its effect on adenylate cyclase.

[³H] -Concanavalin A binding to brain particulate preparations measured by a filtration technique was found to show a characteristic regional specificity with the caudate-putamen area exhibiting the greatest density of concanavalin A (con A) binding sites. The synaptic membranes were shown to be the most highly enriched of the subcellular fractions examined in terms of lectin-binding glycoproteins. Con A was also shown to inhibit the basal adenylate cyclase activity of cerebral, cerebellar, and caudate-putamen particulate preparations in a concentration-dependent manner. This lectin sensitivity of the adenylate cyclase is apparently an intrinsic property of the enzyme complex since a detergent dispersed preparation of the cerebral cortical enzyme was equally inhibited by con A. It is proposed that one of the membrane con A binding sites in brain tissue is a component of the adenylate cyclase system.     

The effects of cyclic AMP on disaggregation-induced changes in activities of developmentally regulated enzymes in Dictyostelium discoideum.

The addition of cyclic AMP to the shaking medium of cells disaggregated from pseudoplasmodia of $\text{Dictyostelium discoideum}$ suppressed the accumulation of cell-bound phosphodiesterase which normally occurs (1) after disaggregation. The suppression was not secondarily brought about by its possible inhibitory effect of cyclic AMP on protein synthesis or by its stimulating effect on the release of the enzyme into the medium. The effect was reversible and specific to cyclic AMP. On the other hand, the inhibitory effect of cyclic AMP on the disaggregation-induced inactivation of UDP-galactose transferase was not apparent in the initial period, but thereafter it slowed down the decrease in the enzyme activity. These results indicate that exogenous cyclic AMP mimics at least in part the regulatory effects of cell-to-cell contact on certain enzymes.     

In vitro studies of skeletal muscle membranes characterization of a phosphorylated intermediate of sarcolemmal (Na+ + K+)ATPase

The sarcolemmal membranes isolated from rat skeletal muscle are capable of incorporating ³²P from [γ?³²P]ATP. The membrane protein phosphorylation requires Mg²⁺. Cyclic AMP, cyclic GMP and their dibutyrul derivatives showed no marked effect on sarcolemmal phosphorylation.The Mg²⁺-dependent ³²P labeling was significantly enhanced by Na⁺. The rate of Na⁺ -stimulated ³²P incorporation was quite rapid reaching steady state levels within 5 s at 0 °C. K⁺ reduced the Na⁺ -stimulated ³²P-incorporation but enhanced the ³²P_i release. This inhibitory effect of K⁺ on Na⁺ -stimulated ³²P incorporation was prevented by the cardiac glycoside, ouabain.The Na⁺ -dependent ³²P labeling showed substrate dependency and the Na⁺ site was saturable. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP was 2 · 10?5 M. The optimum pH for ³²P labeling was between 7 and 8.Na⁺ -dependent membrane phosphorylation showed a direct relationship with the (Na⁺ + K⁺ATPase activity. The high turnover rate of ³²P intermediate (12 000 min ?1) suggested its functional significance in the overall transport ATPase reaction sequence.The predominate portion (> 90%) of the phosphorylated membrane complex was sensitive to acidified hydroxylamine and to alkaline pH suggesting an acylphosphate nature of the phosphoprotein.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ³²P incorporation occurred predominately into a 108 000 dalton subunit which is a major protein component of sarcolemmal membranes. A very low level of ³²P incorporation was also observed into a 25 000 dalton subunit and Ca²⁺ slightly enhanced the phosphorylation of this component.The size ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{108 000}$) and some properties of the sarcolemmal phosphoprotein are closely similar to other (Na⁺ + K⁺ATPase preparations reported so far.     

A fluorogenic method for the demonstration of human serum 5′-nucleotide phosphodiesterase isozymes after polyacrylamide gel electrophoresis

A rapid fluorogenic method for the demonstration of 5′-nucleotide phosphodiesterase in human serum has been developed. This method uses the substrate 4-methylumbelliferyl 5′-thymidylate impregnated in agarose gels or filter paper strips. Zymograms are developed in less than 30 min at 25°C, and the sensitivity of this method has been compared with that of the indigogenic method.     

Preparation of renal cortex basal-lateral and brush border membranes. Localization of adenylate cyclase and guanylate cyclase activities

Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and γ-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na⁺ + K⁺)-ATPase. However, the specific activity of (Na⁺ + K⁺)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na⁺ + K⁺)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na⁺ + K⁺)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5′-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN₃ plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na⁺ + K⁺)-ATPase, be used as an enzyme “marker” for the renal basal-lateral membrane.     

Fluctuations du taux d'AMP cyclique et des activites adenylate cyclase et AMP cyclique-phosphodiesterase dans les cultures synchrones d'un procaryote, Nocardia restricta

Cultures of Nocardia restricta, a prokaryote from the group of Actinomycetes, can be synchronised by diluting, in a fresh growth medium, cells already in stationary phase. The synchronisation of the cultures is monitored by examining the synchrony of DNA replication.In these synchronised cultures, the intracellular cyclic AMP level exhibits rythmic oscillations with a period equal to the generation time of the culture. There is only one peak per generation. The average ratio of maximum to minimum concentrations is at least 3.Cyclic AMP accumulates also in the medium with a step pattern. It appears in the medium during maximum production of cyclic AMP in the cell.The specific activity of adenylate cyclase (EC 4.6.1.1) measured in the 30 000 × g pellet of cell-free extracts also oscillates and correlates well with fluctuations in the cyclic AMP level. At the end of exponential growth, cyclic-AMP phosphodiesterase (EC 3.1.4.17) is detectable in the cells. The specific activity of this enzyme measured in the 30 000 × g supernatant of cell-free extracts shows also an oscillating pattern.To our knowledge it is the first time that such oscillations in the metabolism of cyclic AMP are described among prokaryotes. It is now possible to look at a link between this phenomenon and the cell cycle of the organism.     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.